Biochemistry 0 Copyright 1978 by (he American Chemical Society
Volume 17, Number 24
November 28, 1978
DNA Bifunctional Intercalators. 1. Synthesis and Conformational Properties of an Ethidium Homodimer and of an Acridine Ethidium Heterodimert Bernard Gaugain,i Jacques Barbet,$ Robert Oberlin,t Bernard P. Roques,*-i and Jean-Bernard Le Pecqs
ABSTRACT:
An ethidium homodimer and an acridine ethidium heterodimer have been synthesized. The ethidium and the acridine chromophore were introduced in such bifunctional intercalators in order to allow the fluorometric study of the interaction of such molecules with DNA, which is reported in the companion paper (Gaugain, B., Barbet, J., Capelle, N., Roques, B. P., & Le Pecq, J . B. (1978) Biochemistry 17 (following paper in this issue)). In the preparation of the acri-
dine-ethidium dimer, we report the use of acetyl groups as new protecting agents in the phenanthridine series. Conformational studies of these molecules by visible absorption and N M R spectroscopy indicate that these dimers exist in equilibrium between folded and unfolded conformations and that this equilibrium is pH and temperature dependent. Models for the geometry of the folded forms are proposed.
M a n y DNA intercalating compounds elicit biologically interesting properties and several are used as antitumoral or antiparasitic drugs. It is generally agreed that these properties are related to their reactivity with DNA. In search of more active compounds it is logical to design molecules with the highest possible affinity for DNA. Such a result can be simply obtained using oligomeric derivatives. If each of the subunits could interact with DNA, the binding constant should increase almost exponentially with the number of subunits. Along this line, several bifunctional compounds made up of D N A intercalating dyes (chloroquine, acridine, ellipticine) have recently been prepared (Marquez et al., 1974; Barbet et al., 1975; Canellakis et al., 1976; Delbarre et al., 1976), and several have antitumoral properties (Canellakis et al., 1976; Sinha et al., 1977; unpublished results from our laboratory). Although such molecules can bind to D N A according to different processes, some of them are able to bisintercalate in D N A (Le Pecq et al., 1975; Wakelin et al., 1976). The length and the confor-
mation of the chain linking the two dyes, as well as the chemical nature of the intercalating moieties, have been shown to be of great importance for the interaction with D N A (Le Pecq et al., 1975; Barbet et al., 1976; Canellakis et al., 1976; Fico et al., 1977). Detailed study of the interaction of these types of molecules with D N A is rendered difficult owing to their high binding affinity. Nevertheless, such a study is necessary if one wants to understand the mechanism of action of such compounds. We were therefore led to synthesize new bifunctional derivatives made up of fluorescent compounds such that the D N A interaction could be more easily studied. Ethidium and acridine were chosen for their well-known D N A intercalating and fluorescent properties (Le Pecq, 197 1, 1976, reviews). In this paper we report the synthesis of an ethidium homodimer “EtDi”’ and of an acridine ethidium heterodimer “AcEtDi” (Figure 1) and studies of their conformational behavior as a function of p H and temperature by visible and N M R spectroscopies. The following paper details the use of the fluorescent properties of such compounds for studying the D N A binding of bifunctional intercalators (Gaugain et al., 1978).
t From the Dtpartement de Chimie Organique, associt au C N R S No. 61 3, Universitt RenC Descartes, 4 avenue de I’Observatoire, 75006 Paris, France, and from the Laboratoire de Physicochimie Macromoltculaire LA C N R S No. 147, Institut Gustave-Roussy, 94800 Villejuif, France. Received May 23, 1978. Support of this research through grants from Universitt Rent Descartes (Paris V), Inserm (U147), Universitt Pierre et Marie Curie (Paris VI), DtlCgation i la Recherche Scientifique et Technique, and the Petroleum Research Fund. administered by the American Chemical Society, is gratefully acknowledged. Present address: DCpartement de Chimie Organique, ERA (CNRS) 613, UER des Sciences Pharmaceutiques et Biologiques, 4 avenue de I’Observatoire, Paris, 75006, France. 0 Present address: Laboratoire de Physicochimie Macromoltculaire, LA (CNRS) 147, Institut Gustave-Roussy, 94800 Villejuif, France.
*
0006-2960/78/0417-5071.~01.OO/O
Experimental Section The purity of all the compounds was tested by thin-layer chromatography on silica gel with a 8:2:1 mixture of butanol, I Abbreviations used: EtDi, ethidium dimer; AcEtDi, acridine ethidium dimer; HMDS, hexamethyldisiloxane; MezSO (or DMSO in figure), dimethyl sulfoxide.
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of concentrated hydrochloric acid to yield 3.8-dicarbethoxyamino-5-(4’,8’, 12’-triazadodecyl)-6-phenylphenanthridinium chloride trihydrochloride, VI1 (2.85 g, 66%), mp 235 OC ( R f 0.1). 5- ( 1 I - ( 2-Mel hoxj3- 6 -c h lorn-Y -acridiny laniino ) -4,R-dinY‘ ,I zaundecyl) -3,8-dicarbethoxyamino-6-phenylphenanthridiI I G L : K E I : Cheniical structure of the ethidium honiodirner V I and the niurri Chloride 7’rih!,drochloride, V I I I . 3,X-Dicarbethoxyacridine ethidium heterodimrr IX. amino-5-(4’,8’, I 2’-triazadodecyl)-6-phen) Iphenanthridiriiutn chloride trihydrochloride, VI1 (2.4 g; 3.23 mmol), and 1.3 g trifluoracetic acid, and water as eluent, and their Rj given in (3.88 mmol) of 2-methoxy-6-chloro-9-phenoxyacridinewere brackets. heated under stirring at 120 OC in 22 g of phenol for 1.5 h. On cooling the mixture solidified. It was dissolved in 11 m L of I . Synthesi~ methanol and the brown solution slowly poured into 360 mL 2.8-Dirarbethoxyamino-5- (4’,7’-diazaheptyl) -6-phenylof ether to precipitate 5-( I 1 - ( 2-methox~-6-chloro-9-acriphenanthridiriiiirn Chloride Dihydrochloride, IV. 3,8-Didin y 1 a mi no) - 4.8 - d i a 7a II nd ec y 1) ..3.8 -d i c;i r be t h o x ),am i n 0- 6 carbethox~amino-5-(3’-bromopropyl)-6-phenylphenanthriphenylphenanthridinium chloride trihydrochloride. VI1 I, diniuni bromide, 111 (9.6 g, 15.2 mmol) (Watkins, 1952), was which crystallized from methanol in orange crjstals (2.3 g, suspended in 570 mL of methanol. By addition of 20 mL (0.3 73%), mp 272 OC (R, 0.2). mol) of ethylenediamine, the foregoing salt was dissolved and 5 - ( I 1 (2 - Met hoxy - 6 - c 11loro - 9 - acrid i ny la rri i no ) - 4,8- d ia the solution was refluxed for 6 h. The cooled solution was zaundecyl)-3,8-dianiint~-6-phenylphenanthridinium Chloride poured into 75 m L of water. Evaporation of methanol gave Pentahydrochloride. IX’ I- AeEtDi (Method ,1).The protected orange plates which were washed with water then dried. This heterodimer, V l l I (279 mg: 0.28 mmol), was heated under pseudo-base was dissolved in 700 m L of ethanol and the solustirring with 0.5 rnt. of concentrated sulfuric acid for 3 h. The tion acidified by 6.3 mL of concentrated hydrochloric acid to cooled mixture \\as made basic by the addition o f 2.2 ml. of yield 3,8-dicarbethoxyamino-5-(4’,7’-diazaheptyl)-6-phenyl I O N ammonia solution, yielding a “brown gum,” which was phenanthridinium chloride dihydrochloride, IV (5.2 g, 78%). separated and dissolved in concentrated HCI. This solution was mp 278 O C (RJ 0.60). then chromatographed on a column of silica gel, with an 8:2:1 Anal. Calcd for C30H38Cl3N504-lH~O:C, 54.84; H, 6.14; mixture of butanol, trifluoroacetic acid, and water as eluent. CI, 16.19; N , 10.66; 0, 12.18. Found: C, 54.88; H, 5.79; C1, The resulting product ( R , 0.1 by thin-layer chromatography 16.25; N, 1 I .05; 0, 1 1.88. on silica gel using above eluent) was evaporated to dryness and 4,7-Diazadec~~l-5,5’-his(3,8-dicarbetho.xyamino-6-hydissolved in concentrated HCI. The heterodimer, IX, was then droxy-6-phertyl-5,6-dihydrophenanthridine), V. Five separated as a red oil, by adding acetone to the above mixture. grams (7.85 mmol) of 3.8-dicarbethoxyamino-5-(4’,7’-dia- Subsequent addition of ether to this oil produced a solid product, IX, which was filtered and dried in vacuo (30 mg, zaheptyl)-6-phenylphenanthridinium chloride dihydrochloride, IV, was suspended in 250 ml, of methanol; then the mixture 12%). mp 265 “ C ( R , 0. I ). was boiled. Sixteen milliliter of 1 N N a O H was added. The Anal. Calcd for C42HiuC1,N70.41-Iz0:C, 5 1 .OO; H, 5.91; orange solution obtained was treated with I6 mL of 1 N N a O H CI, 25.09; N, 9.91; 0,8.09. Found: C, 51.13; H and 5 g (7.9 mmol) of 3,8-dicarbethoxyamino-5-(3’-bromo- N,9.83: 0, 8.63. 3,8-Diacetanrido-6-pheiiylphenanthridine, X.Acetic anpropyl)-6-phenylphenanthridinium bromide, 111, dissolved in hydride (2.5 mL) was added to 855 rng (3 mmol) of 3,8-di200 mI. of methanol, was added to the solution mixture. After 16 h of refluxing, the solution was cooled and the protected amino-6-phenylphenanthridine, I (Watkins, 1952), in 5 ml, ethidium dimer crystallized as the pseudo-base, V, in yellow of acetic acid. The mixture was stirred a t room temperature for 0.5 h and 3,8-diacetamido-6-phenylphenanthridiniuni plates. It was washed with methanol then dried (2. I 5 g, 26%; Rj 0.55). acetate precipitated. The suspension was slowly poured into 20 mL of water, and then made basic with I 5 mL of concen4,7-Diazadecyl-5,5’-bis(3,8-diamino-6-p~7enylphenantrated ammonia solution to yield the phenanthridine. It was thridium) Dichloride Dihydrochloride, VI = Ethidium washed with water, and then dried and crystallized from ethMoniodimer, EtDi.The pseudo-base, V (2.1 5 g; 2.06 nimol) wac hjdroly7ed by heating with 4.8 mL of concentrated sulanol in very pale yellow crqitals (945 mg, 86%), nip 205 “C ( R f furic acid at 130 “c‘.The cooled mixture was made basic with O.SC), 19 mL of I O N ammonia solution to liberate a crude pseudoAnal. Calcd for C23f1,9N302.lH~O: C:, 71.30; H, 5.46; N. base. It was dissolved i n 7.5 m L of concentrated hydrochloric 10.85: 0, 12.39. Found: c‘. 71.99; H. 5.28; N , 11.02; 0 , acid. By addition of 15 mL of acetone, the ethidium dimer 11.32. 3,X-Diacetuniido-5- (3‘-hrornopropyl)-6-pheti!.lphenancrystalli7ed in purple crystals ( I .45 g, 96%), mp 275 O C . Anal. Calcd for C46H5oCI~N8.2CH30FI.5H20: C, 57.03; thridinium Bromide, XI. 3,8-Diacetamido-6-phenylphenant i , 6.78; CI, 14.03; N. 11.08; 0, 11.08. Found: C, 57.67; H. thridine, X (500 mg; 1.36 rnmol), was refluxed with 50 mL of 5.68;CI, 14.08;N. 11.22;0, 11.2O(Rj0.09). 1,3-dibromopropane for 6 h. Yellow plates were deposited. After cooling, the mixture was filtered and 3,8-diacetamido3,8-Dicarbethoxyamino-5- (4‘,8’, 12’-triazadodecy1)-6pheri)~lpherianthridiuni C‘hloride 7’rihydrochloride. VTI. 5-(3’-brornopropyl)-6-phenylphenanthridiniumbromide, XI, Bis(3-aminopropy1)amine (7.4 mL; 50.9 mmol) was added to crystallized from methanol in yellow plates (615 mg, 809/0), mp a suspension of 3.7 g (5.85 mmol) of 3,8-dicarbethoxyamino280 OC (Rr 0.45). 5-(3’-bromopropyl)-6-phenylphenanthridinium bromide, 111, Aria). dalcd for CZ6H2sRrZN302.1CH30H: C, 53.75: 1-1, in 7.5 mL of methanol. After 5 h of refluxing, the red solution 4.84: Br. 26.49: N. 6.96; 0. 7.96. Found: C, 53.05; €3, 4.62; Br, was cooled arid poured into water (25 mL). Then methanol was 26.47: N . 6.80; 0. 6.10 evaporated in vacuo to precipitate the pseudo-base which was 3.8-Diacetamido-5- (4’,8’, 12’-triazadodecyl)-6-phenylphenanthridinium Chloride Trihydrochloride, XII. 3,8-Diwashed with water then dried. The orange plates were dissolved acetamido-5-( 3’-bromopropyl)-6-phenylphenanthridinium in I O mL of ethanol and the solution was acidified with 1.6 mL ~
DNA BIFIJNCTIONAI. 1NTERCAL.ATORS
1. SYNTHESIS
bromide, XI (500 mg; 0.88 mmol), was stirred under reflux with 4 m L (28.4 mmol) of bis(3-aniinopropyl)aminein 15 mL of methanol for 6 h. The cooled solution was poured into water and methanol evaporated. A yellow solid was deposited. It was washed with water then dried. This pseudo-base was dissolved in warm ethanol. By acidification with 10 N hydrochloric acid, 3,8-diacetamido-5-(4'-8',12'-triazadodecyl)-6-phenylphenanthridinium chloride trihydrochloride, XI], precipitated. It was crystallized from methanol as orange plates (385 mg, 64%), mp 252 "C (RJ0.05). Anal. Calcd for C32H44C14N6O2-5H2O:C, 49.49; H , 7.01; CI, 18.26; N , 10.82; 0, 14.42. Found: C, 48.80; H, 6.34; CI, 17.88; N, 10.28; 0, 14.05. 5 - ( I 1 - (2-Methoxy-6-chloro-Y-acridinylamino)-4,8-dia-
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zaundecyl)-3,8-diacetamido-6-phenylphenanthridinium Chloride Trihydrorhloride, X I I I . 3,8-Diacetamido-5-(4', 8',12'-triazadodecyl)-6-phenylphenanthridinium chloride trihydrochloride, XI1 (274 mg; 0.4 mmol), and 161 mg (0.48 mmol) of 2-methoxy-6-chloro-9-phenoxyacridine were heated under stirring a t 120 "C in 1 g of phenol for 2 h. On cooling the mixture solidified. After dissolution in 2 m L of methanol, the resulting brown solution was poured into 100 m L of ether. 5 4 1 1-(2-Methoxy-6-chloro-9-acridinylamino)-4,8-dia-
VI
FIGURE 2:
Scheme of EtDi synthesis.
measured by spectrophotometric analysis. For each study and zaundecyl)-3,8-diacetamido-6-phenylphenanthridinium a t each concentration, all the IH signals were assigned from chloride trihydrochloride, XIII, precipitated. It was then fildouble resonance experiments. tered, washed with ether, and crystallized from methanol in brown-orange prisms (327 mg, 88%), m p 260 "C (R, 0.15). 3. Visible Spectra Anal. Calcd for C46H52CI5N7O3-4H20: C, 55.23; H, 6.05; Visible spectra of AcEtDi and of the parent compounds XIV CI, 17.72; N , 9.80; 0, 11.20. Found: C, 55.31; H , 6.15; CI, and XV have been recorded on a Varian Techtron 635 UV16.29; N, 9.21; 0, 11.22. M in 0.1 M ac5 - ( I I -( 2-Methoxy-6-chloro-9-acridinylamino)-4,8-dia- visible spectrometer a t concentration of etate buffer (pH 5 ) . Studies of the AcEtDi and EtDi visible zaundecyl)-3,8-diamino-6-phenylphenanthridinium Chloride spectra as a function of p H were monitored a t 5 X M (25 Pentahydrochloride,IX = AcEtDi (Method B). 5-(11-(2Methoxy-6-chloro-9-acridinylamino)-4,8-diazaundecyl)- "C) in 0.05 M NaCl solutions. 3,8-diacetamido-6-phenylphenanthridiniunichloride trihyResults and Discussion drochloride, XI11 (562 mg; 0.61 mmol), was suspended in 28 m L of 10% HCI-methanol. The mixture was refluxed under I. Synthesis stirring for 2 h. The cooled solution was filtered; then the solvents were removed by evaporation in vacuo for 4 h to give 1. Ethidium Homodimer "EtDi" Vl. 4,7-Diazadecyl5-( 1 1-(2-methoxy-6-chloro-9-acridinylamino)-4,8-diazaun- 5,5'-bis(3,8-diamino-6-phenylphenanthridinium) dichloride decyl)-3,8-diamino-6-phenylphenanthridinium chloride dihydrochloride, VI (ethidium homodimer "EtDi"), has been pentahydrochloride, IX, as a purple power (436 mg, 79%), mp synthesized according to the scheme in Figure 2; 3,8-di265 "C (RJ0.1). carbethoxyamino-5-( 3'-bromopropyl)-6-phenylphenanthriAnal. Calcd for C42H&17N70.4H20: C, 51.00; H , 5.91; dinium bromide, 111, has been prepared according to Watkins CI, 25.09; N, 9.91; 0,8.09. Found: C, 50.84; H , 5.78; CI, 23.42; (1 952). By reaction with ethylenediamine, this quaternary salt N , 10.00; 0, 8.55. gives 3,8-dicarbethoxyamino-5-(4',7'-diazaheptyl)-6-phenylphenanthridinium chloride dihydrochloride, 1V. This com2. N M R Studies pound is allowed to react with the bromopropyl quaternary 'H N M R spectra were recorded at 270 M H z with a Bruker compound 111 leading to 4,7-diazadecyl-5,5'-bis(3,8-diW H 270 spectrometer operating on the Fourier transform carbethoxyamino-6'- hydroxy-6-phenyl -5,6-dihydrophenanmode and locked to the deuterium resonance of solvent DzO. thridine), V, the pseudo-base of the protected ethidium Probe temperatures were regulated by a Bruker B ST 100/700 homodimer. This intermediate is not purified but is immedicontroller and monitored by observing the splitting in ethylene ately reacted with sulfuric acid. The protective carbethoxy glycol. Chemical shifts (6) were measured from an external groups are then hydrolyzed. After neutralization, the ethidium reference made up of a capillary filled with a solution of 5% homodimer VI is crystallized. The structure of EtDi has been hexamethyldisiloxane (HMDS) in CCI4. 6 in ppm are reliable established previously, by N M R experiments (Roques et al., to fO.O1 ppm. 1976). In order to reduce the intensity of the HOD solvent resoAttempts to synthesize other ethidium homodimers, with nance, the spectra, a t concentration M, were recorded different chain lengths, have still been unsuccessful. Attempts with a strong and continuous irradiation of the H O D signal. for nucleophilic displacement of the bromine atom of 111 by For lower concentrations, standard homonuclear gated decompounds similar to IV but with longer alkylamino chains coupling has been used. have led to decomposition with the recovering of some pheSolutions were made in pure D 2 0 at different pD values nylphenanthridine. On the other hand, the dimer cannot be which were determined with a Tacussel TC 60/N p H meter obtained directly by reaction of an excess of I11 with the diaccording to the equation, pD = meter reading 0.4 (Glasoe amine as for the preparation of acridine dimers (Barbet et al., & Long, 1960). The concentrations of the stock solutions were 1975).
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XIV
F I C L R E 4:
3,8-Diamino-5-(3'-diethylaminopropyl)-6-phenylphenanthridinium iodide hydroiodide. XIV, and 2-methoxy-6-chloro-9-(3'diniethy1aminopropylamino)acridiniumchloride hydrochloride, XV. used as parent compounds in visible and N M R studies.
VI11 R : C q E t
xlll
F I G U R E 3:
R:COCk$
Scheme of AcEtDi synthesis.
2. Acridine Ethidium Heterodimer "AcEtDi". 5 4 1 1 -(2Methoxy-6-chloro-9-aminoacridinyl)-4,8-diazaundecyl)3,8-diamino-6-phenylphenanthridiniumchloride pentahydrochloride, IX, has been synthesized according to the scheme presented in Figure 3; 3,8-diamino-6-phenylphenanthridine, 1, prepared according to Watkins (1952) reacts with acetic anhydride to give 3,8-diacetamido-6-phenylphenanthridine, X, which is quaternized by 1,3-dibromopropane: 3,8-diacetamido-5-(3'-bromopropyl)-6-phenylphenanthridinium bromide, XI, thus obtained is allowed to react with an excess of bis(3-aminopropy1)amine to give 3,8-diacetamido-5(4',8', 12'-triazadodecyl)-6-phenylphenanthridinium chloride trihydrochloride, XII. This compound reacts with 2methoxy-6-chloro-9-phenoxyacridineto afford the protected AcEtDi, XIII. When refluxed in methanol-HCI according to Walls & Whittaker (l952), the acetamido groups are rapidly hydrolyzed and AcEtDi is obtained in good yield and can be used without further purification. The best way for the synthesis of such an heterodimer AcEtDi is to start from the aminoalkylphenanthridinium XI1 according to the above scheme (Figure 3). We have attempted, alternatively, to effect the heterodimer synthesis beginning with aminoalkylacridine monomers, which can be prepared by use of a considerable excess of polyamine. However, spontaneous dimerization occurs both during purification and during storage a t room temperature. All attempts to make these compounds react with halogenoalkyl derivatives have failed and the corresponding acridine homodimer is obtained as the major product. As for EtDi, preparation of AcEtDi requires the previous protection of ethidium amino groups. In a first assay, the carbethoxy group has been used like a protective agent as described by Watkins (1952) and Walls (1947). Removal of these
Wavelength
(nm)
5 : Visible spectra in 0.1 M acetate buffer (pH 5) of (a) AcEtDi, (b) phenanthridinium compounds XIV, and (c) acridinium derivative XV. Concentration \+as9.0 X IO-' M for each compound. FIGURE
groups requires the use of hot concentrated sulfuric acid. Because of the sensitivity of the 9-aminoacridine toward strong acidic or alkaline medium (Albert & Ritchie, 1943), in this method, the yield of the last step is poor (12%). Therefore, more easily removable protective agents must be used. Unfortunately, protection of the amino groups of the phenanthridine I with formyl or carbobenzyloxy groups inhibits the quaternization by 1,3-dibromopropane. However, a good yield is obtained with acetyl protecting groups removable in relatively mild conditions (Walls & Whittaker, 1952). Thestructure of AcEtDi has been established by visible and N M R experiments. The visible spectrum of AcEtDi was compared with those of the parent compounds: 3,8-diamino5-(3'-diethylaminopropyl)-6-phenylphenanthridinium iodide hydroiodide, XIV, and 2-methoxy-6-chloro-9-(3'-diethylaminopropy1amino)acridinium chloride hydrochloride, X V (Figure 4). Such compounds XIV and XV containing an aminoalkyl chain are more suitable, because it has been shown that substitution by charged chains appreciably modifies the spectroscopic properties of the chromophore (Le Bret & Chalvet, 1977). Visible spectrum of AcEtDi (Figure 5 ) exhibits all the characteristic absorption bands of XIV and XV. The N M R spectrum of AcEtDi in MezSO-dh solution is reported in Figure 6. Assignment of all resonances was made by comparison with XIV and XV and with the aid of double-resonance experiments. The ' H N M R spectrum of XV has been previously interpreted (Barbet et al., 1976).
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IH N M R spectrum of the acridine ethidium heterodimer at 20 OC (lob2 M) in MezSO-db; H M D S was used as an internal reference.
Visible spectrum of ethidium dimer (0.5 X M) in 0.05 M NaCl for different values of pH. (-) pH 3.0; (- - -) pH 4.2; (- -) pH 7.4. pH 4.9; (-- -)pH 5.8; F I G U R E 7:
(.e*)
I I
.
9 H I
20
40
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11. Conformational Studies of EtDi and AcEtDi
FlGURE 8: Temperature dependence of the chemical shifts of the aromatic protons of EtDi M) as compared with those of phenanthridinium monomer XIV M), at pH 7 . A6 = 6 x 1 -~ 6EtDi.
Similar to acridine dimers (Barbet et al., 1976), both EtDi and AcEtDi can exist in equilibrium between folded and unfolded conformations. Such behavior could be of great importance for the DNA intercalation process. Before any DNA interaction study, it was necessary to analyze the conformational states of the present dimers in aqueous solution at different pH. 1 . Ethidium Dimer. Since the ethidium dimer is a bisquaternary ammonium compound, its ionization will not be strongly affected by varying the pH of its aqueous solution. However, the visible spectrum of EtDi shows a red shift as a function of pH and also hypochromism (Figure 7 ) , whereas no such behavior is observed in the phenanthridinium monomer XIV. This can be easily explained by the titration of the ammonium charges of the chain linking the two rings, which decreases the electrostatic repulsion between the ammonium groups of the chain and the quaternized rings. This effect leads to an important increase of self-stacked dimers in accordance with the observed hypochromism. Indeed, Warshaw & Tinoco (1 966) have shown that strong stacking interactions, as in dinucleotides, generally lead to hypochromism. In order to confirm the presence of such intramolecular stacked structures, we have performed N M R studies in M D 2 0 solutions. At this concentration, intermolecular interactions both for ethidium monomer and dimer are minimized as already reported (Roques et al., 1976). At 21 O C and pH 7, a comparison of the N M R spectra of EtDi and the cor-
responding monomer XIV shows a strong upfield shift for the H I , Hz, H4, and Hlo protons in the dimer (Figure 8). This shielding effect is about twice that observed at pH 3. With respect to these results, it can be concluded that EtDi is in equilibrium between folded and unfolded conformations in accordance with the results of the visible study. This equilibrium can be monitored as a function of temperature between 21 and 80 OC at pH 7 where the folded form is expected to be preponderant (Figure 8). On raising the temperature, a marked deshielding is observed for the H I , Hz, H4, and Hlo protons of EtDi as compared with similar protons of XIV in the same conditions. In contrast, the less shielded protons H7 and Hg are practically unaffected. Because the strongest shielding is observed for the H I , H2, Hq, and H I Oprotons as compared with the H7 and Hg protons, a model for the folded form (Figure 8) can be proposed using ring current calculations made by Giessner-Prettre & Pullman (personal communication) on ethidium bromide. 2. Acridine Ethidium Dimer. As for EtDi, the visible spectrum of the acridine ethidium dimer shows an important hypochromism on the acridine band on raising the pH, whereas a moderate hypochromism and a weak red shift appear for the phenanthridinium band (Figure 9). The curves of the absorption intensity as a function of pH at fixed wavelengths clearly shows the presence of two titrations (Figure 10). The first titration (low pH) can be monitored by changes in either
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F l G L R E IO:
Absorption intensity of acridine ethidium dimer (0.5 X loT4 (-m-)and X = 480 nm (-0.)as a
Visible spectrum of acridine ethidium dimer (0.5 X M) in 0.05 M NaCl for different values of pH. (--) pH 3.0; (- - -) pH 5.1: ( ~~- ---) pH 6.0: (- . -) pH 6.6; ( * . .) pH 8.0: (- . . --) pH 9.5.
M) in 0.05 M NaCl at X = 440 nm function of pH.
the acridine or the phenanthridinium absorption bands, whereas the second titration is observed only when monitoring changes in the acridine absorption. The first titration (pK 6.0) probably corresponds to successive deprotonations of the ammonium charges on the chain linking the two rings, leading to an increase of the stacked dimer population (similar to that observed for EtDi). The second titration (pK = 7.9) is easily identified as the deprotonation of the intracyclic nitrogen atom of the acridine. This is readily apparent by comparison with the visible spectrum of the acridine monomer XV, in which the pK of the intracyclic nitrogen is found equal to 8.2. Due to the overlap of the acridinium and phenanthridinium chromophores in the visible spectrum, we have further investigated the pH dependence of the AcEtDi stacking interaction by N M R . The assignments of the resonances of all the protons, except HI,, H3!, and H41 which are superimposed, were established by comparison with XIV and XV and double-resonance experiments (Figure 1 1 ) . The methylene signals of the aminoalkyl chain a t 4.1 ppm (a) and 3.9 ppm (k) have been assigned to the CH2 linked to the phenanthridinium ring and the 9-acridine amino group, respectively, in accordance with their position in the plane of the aromatic ring and the vicinity of the charged phenanthridinium nitrogen for the former. On the other hand, the more shielded signal a t 1.8 ppm has been attributed to the CH2 group (f) located a t the center of the chain and far from the two aromatic rings. From these assignments, all of the other CH2 signals have been assigned by double-resonance (Figure 1 1). At pH 7,21 “C, and a t low AcEtDi concentration (5 X M), several of the aromatic protons ( H I , H2, H4, H,, Hs,, and Hg,) of AcEtDi are observed shifted upfield by 0.3 to 0.6 ppm, as compared with identical protons in the N M R spectra of the monomeric compounds XIV and XV. The induced shielding of these protons on AcEtDi readily suggest the presence of some folded structure in the dimer. It must be emphasized that at this concentration ( to M AcEtDi), intermolecular dimer-dimer interactions are negligible as observed in previous N M R studies of EtDi (Roques et al., 1976). Furthermore, the aromatic resonances which are shielded in the AcEtDi N M R spectrum at pH 7 and a t 21 O C ( H I , H2, H4, H7, H s ~and , Hg,) undergo slight downfield shifts (60.1 ppm; data not shown) as the temperature is raised to SO O C , providing further evidence for the presence of a folded + unfolded dimer equilibrium in solution. However, the small magnitude of these temperature induced shifts (GO.1 ppm) suggests that a folded configuration still predominates at pH 7, even a t higher temperatures. Variations in the chemical shifts of all the assigned protons of AcEtDi at M are presented as a function of p H in Figure 12. Pronounced upfield shifts of 0.1-0.5 ppm are ob-
served upon increasing pH, for protons on the methylene chain (Figure 12A) and on the two aromatic chromophores (Figure 12B). These results are consistent with a folded dimer conformation a t high pH, which results in increased proton shielding due to stacking of the two aromatic chromophores. The shapes of the titration curves (Figure 12) indicate that the largest induced chemical shift changes occur between p H 5.5 and pH 9. Deprotonation of both the acridine ring (pK 8, Figure 10) and the chain amino groups (pK 6, Figure 10) directly influences the intramolecular stacking interactions between aromatic rings, as observed by the induced chemical shift changes of the AcEtDi protons (Figure 12). On the phenanthridinium ring, the largest induced upfield shifts with increasing p H (pH 5.5-9) are observed for the H I , Hz, H4, and H7 protons (A6 = 0.3-0.4 ppm), whereas the Hlo and Hg protons are shifted upfield by only 0.17 and 0.06 ppm, respectively. On the acridinium ring, the resonances from the methoxy group and those of the HI’, H3!, and H4’ protons (data not shown) are weakly shielded (A6 = 0.05 ppm), whereas the Hgl, H7‘. and Hs. proton resonances are strongly upfield shifted by 0.3-0.4 ppm (Figure 12B). This internal comparison between protons located on each side of the two heterocyclic moieties leads us to propose a folded conformation for AcEtDi with partial overlap between the two chromophores, analogous to that proposed for EtDi. In such a model, the portion of the acridine ring carrying the methoxy group would not be stacked over the phenanthridinium ring (in agreement with the above induced chemical shift results), thus decreasing steric hindrance with the 6-phenyl substituent.
I IGLRL: 9:
-
-
-
Conclusion Two bifunctional intercalators EtDi and AcEtDi made up of highly fluorescent heterocycles (phenanthridine and acridine) have been synthesized. In the series of acridine dimers, we have shown (Barbet et al., 1976) that such bifunctional compounds are able to bisintercalate in DNA by the so called “excluded site” model (Bauer & Vinograd, 1970). Such a binding process requires that the distance between the two aromatic rings is >10.1 A. In EtDi and AcEtDi the lengths of the chains linking the two chromophores, computed from model building data, are 11.4 A for EtDi and 13.5 A for AcEtDi. Despite this apparent agreement with the required distance, EtDi and AcEtDi do not bisintercalate in D N A (Gaugain et al., 1978). However, it can be observed that, for the present dimers, these maximal distances between the two rings are calculated with the linking chains in their more extended conformations. Due to the presence of the 6-phenyl ring on the phenanthridinium part of these dimers, such extended forms of the chains are unfavorable because this configuration enhances the steric hindrance between the phenyl ring and several
DNA BIFUNCTIONAL
INTERCALATORS.
1. SYNTHESIS
n
8
FIGURE 1 I :
VOL.
E
5
NO.
24, 1978
5077
I
I
7
17,
4
3,
2
M) in D20 at pH 3.
' H N M R spectrum of AcEtDi
for these folded forms are in accordance with a diagonal orientation of the connecting chain and partial overlap of the aromatic chromophores. It is of considerable importance to recognize that the DNA binding of either EtDi or AcEtDi by a bisintercalation process will require opening of the folded drug forms (Le Pecq et al., 1975). Therefore, the DNA binding free energy of these bifunctional drugs is directly dependent on the strength of their intramolecular stacking interactions. Consequently, it appears that at physiological conditions (pH 7 , 37 "C) the most appropriate bifunctional compounds should be those which retain the highest proportion of unfolded forms in order to exhibit stronger pharmacological DNA-binding properties. Such assumptions have been confirmed in preliminary experiments on these and other derivatives synthesized in our laboratories.
2
PPI
-
I 3
s
7
9
p
H
,
, 3
,
I
I
,
I
i
FIGURE 12: Induced chemical shift changes of the AcEtDi protons as a function of pH.
,
I
9 p H
M)
methylene groups of the chain decreasing their degree of freedom. In order to reduce such an unfavorable entropy factor, the extended conformation of the linking chain must adopt a more diagonal orientation to join the two chromophores. Such a structural requirement decreases the maximal distance between the two aromatic rings and so prevents their DNA bisintercalation (Gaugain et al., 1978). In aqueous solutions EtDi and AcEtDi are in equilibrium between folded and unfolded conformations. As shown by visible and N M R studies, this equilibrium is pH and temperature dependent. However, in comparison with the intramolecular stacking of the acridine dimers (Barbet et al., 1976), the stacked conformations of EtDi and AcEtDi still predominate even at high temperature (80 OC, pH 7) owing to the greater stability of their folded forms. The proposed geometry
Acknowledgments The authors are very grateful to Dr. C. G. Reinhardt for helpful discussions and critical evaluation of the manuscript. References Albert, A., & Ritchie, B. (1943) J . Chem. Soc., 458. Barbet, J., Roques, B. P., & Le Pecq, J. B. (1 975) C. R. Acad. Sci., Ser. D, 8 5 1. Barbet, J., Roques, B. P., Combrisson, S., & Le Pecq, J. B. (1 976) Biochemistry 15, 2642. Bauer, W., & Vinograd, J. (1970) J . Mol. Biol. 47, 419. Canellakis, E. S.,Schaw, Y . H., Hanners, W. E., & Schwartz, R. A. (1976) Biochim. Biophys. Acta 418, 277. Delbarre, A., Roques, B. P., & Le Pecq, J. B. (1976) C. R . Acad. Sci., Ser. D. 284, 81. Fico, R. M., Teh-Kuei-Chen, & Canellakis, E. S. (1977) Science 198, 5 3 . Gaugain, B., Barbet, J., Capelle, N., Roques, B. P., & Le Pecq, J. B. (1978) Biochemistry 17 (following paper in this
5078
B Io c H E M I S T R Y
issue). Glasoe, P. K., & Long, F. A. (1960) J . Phys. Chem. 64, 188. Le Bret, M., & Chalvet, 0. (1977) J . Mol. Struct. 37, 299. Le Pecq, J. B. (1971) in Methods of Biochemical Analysis (Glick, D., Ed.) Vol. 20, pp 41-86, Wiley, New York, N.Y. Le Pecq, J. B. (1976) in Biochemical Fluorescence Concepts (Chen, R. F., & Edelhoch, H., Eds.) Vol. 11, pp 71 1-736, Marcel Dekker, New York, N.Y. Le Pecq, J. B., Le Bret, M., Barbet, J., & Roques, B. P. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 291 5 . Marquez, V. E., Cranston, J. W., Ruddon, R. W., & Burck-
G A U G A I Y
ET AI..
halter, J . H. (1974) J . Med. Chem. 17, 856. Roques, B. P., Barbet, J., & Le Pecq, J. B. ( 1 976) C. R. Acad. Sci., Ser. D 283, 1453. Sinha, B. K., Philen, R. M . , Sato, R., & Cysyk, R . L. ( 1 977) J . Med. Chem. 20, 1528. Wakelin, L. P. G., Romanos, M., Canellakis, E. S., & Waring, M. J . (1976) Stud. Biophys. 60, 1 1 1. Walls, L. P. (1 947) J . Chem. Soc., 67. Walls, L. P., & Whittaker, N. (1952) U S . 2, 594,265, April 22. Warshaw, M. M., & Tinoco, J., Jr. (1966) J . Mol. B i d . 20, 29. Watkins, T. I . (1952) J . Chem. Soc., 3059.
DNA Bifunctional Intercalators. 2. Fluorescence Properties and DNA Binding Interaction of an Ethidium Homodimer and an Acridine Ethidium Heterodimer? Bernard Gaugain,t Jacques Barbet,’ Nicole Capelle,§ Bernard P. Roques,t and Jean-Bernard Le Pecq*,§
Appendix: Numerical Solution of McGhee and von Hippel Equations for Competing Ligands Marc Le Bret#
ABSTRACT: An ethidium homodimer and an acridine ethidium
heterodimer have been synthesized (Gaugain, B., Barbet, J . , Oberlin, R., Roques, B. P., & Le Pecq, J. 9. (1978) Biochemistry 17 (preceding paper in this issue)). The binding of these molecules to DNA has been studied. We show that these dimers intercalate only one of their chromophores in DNA. At high salt concentration (Na+ > 1 M) only a single type of DNA-binding site exists. Binding affinity constants can then be measured directly using the M c Ghee & Von Hippel treatment (Mc Ghee, J. D., & Von Hippel, P. H. (1974) J . Mol. B i d . 86, 469). In these conditions the dimers cover four base pairs when bound to DNA. Binding affinities have been deduced from competition experiments in 0.2 M Na+ and are in agreement with the extrapolated values determined from direct DNA-binding measurements at high ionic strength. As
T h e biological properties and the antitumoral activity of bifunctional intercalators (Fico et al., 1977; Sinha et al., 1977; unpublished results from our laboratory) are thought to be related to their high D N A binding affinity (Le Pecq et al., 1975). It is therefore of interest to characterize and understand in detail the DNA binding of such molecules. Their interaction
expected, the intrinsic binding constant of these dimers is considerably larger than the affinity of the monomer (ethidium dimer K = 2 X lo8 M-I; ethidium bromide K = 1.5 X IO5 M-’ in 0.2 M Na+). The fluorescence properties of these molecules have also been studied. The efficiency of the energy transfer from the acridine to the phenanthridinium chromophore, in the acridine ethidium heterodimer when bound to DNA, depends on the square of the AT base pair content. The large increase of fluorescence on binding to DNA combined with a high affinity constant for nucleic acids makes these molecules extremely useful as nucleic acid fluorescent probes. In particular, such molecules can be used in competition experiments to determine the DNA binding constant of ligands of high binding affinity such as bifunctional intercalators.
with D N A is complex. Several different types of binding are involved and in some cases bisintercalation occurs (Le Pecq et al., 1975; Wakelin et al., 1976). Because the DNA binding affinity of such molecules is very high, a direct measurement of this binding affinity is particFrom the Dtpartement de Chimie Organique, associt au C N R S No. 61 3, Universitt Rent Descartes, Macromoltculaire, 4 avenue de I’Ob-
*
Present address: Dtpartement de Chimie Organique ERA (CNRS) 6 13, UER des Sciences Pharmaceutiques et Biologiques, 4 avenue de I’Observatoire, Paris, 75006, France. 5 Present address: Laboratoire de Physicochimie Macromoltculaire (LA C N R S No. 147), lnstitut Gustave-Roussy, 94800 Villejuif. France. # Laboratoire de Physicochimie Macromoltculaire (LA C N R S 147), lnstitut Gustave-Roussy, 94800 Villejuif, France.
0006-~960/78/0417-5078$01.OO/O
servatoire, 75006 Paris, France, and from the Laboratoire de Physicochimie Macromoltculaire LA C N R S No. 147, lnstitut Gustave-Roussy, 94800 Villejuif, France. Receiued May 23, 1978. Support of this research through grants from Universitt Rent Descartes (Paris V ) , l N S E R M (U 147), Universitt Pierre et Marie Curie (Paris VI), Dtltgration a la Recherche Scientifique et Technique, and the Petroleum Research Fund, administered by the American Chemical Society, is gratefully acknowledged.
0 1978 American Chemical Society
